The global problem of antimicrobial resistance is well recognized by national authorities and rated as a major threat of the 21st century (for a review see Boucher H. et al., Clinical Infectious Diseases 2009, 48(1), 1-12). In contrast to de novo antibiotic development, efforts on alternative approaches such as antibiotic potentiators—following the Augmentin paradigm—have been intensified. The pathogens that cause major problems especially in hospital environments are often summarized as ESKAPE group of opportunistic bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species (Rice L., The Journal of Infectious Diseases 2008, 197(8), 1079-1081), which needs to be expanded with Clostridium difficile. Multi-resistant or extensively drug resistant isolates belonging to this group are increasingly hard to treat, and there exists a raising number of untreatable strains. The only limiting factor in treatment of these pathogens with the existing portfolio of antibiotics is the antimicrobial resistance. Antibiotics that have been prescribed by physicians for decades due to their preferable efficacy or toxicity profile thus had to be largely replaced.
Tetracyclines are proven antibacterial agents and represent one of the most trusted classes of antibiotics. Discovered in the 1940s, the tetracyclines are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site (Griffin M. O. et al., American Journal of Physiology. Cell Physiology 2010, 299(3), C539-48).
The tetracycline class of antibiotics comprises a distinct family of substituted tetracyclic hydronaphthalene compounds produced by strains of Streptomyces aureofaciens and Streptomyces rimosus. First generation tetracyclines such as tetracycline, chlortetracycline, oxytetracycline, and demeclocycline are obtained by biosynthesis. Second generation tetracyclines such as doxycycline, lymecycline, meclocycline, methacycline, minocycline, and rolitetracycline are derivatives obtained by semi-synthesis. Third generation tetracyclines such as tigecycline (Tygacil® Pfizer), pentacycline antibacterials (a structural modification of doxycycline with five rings), azatetracyclines and fluorocyclines (heteroatoms inserted into the D ring), and alkylaminotetracycline antibacterials, are obtained by total synthesis. Although some researchers consider tigecycline to be distinct from other tetracyclines drugs and representing a new family of antibacterials called glycylcyclines, these glycylcyclines are considered tetracyclines for the purpose of the present invention.
Tetracyclines display broad spectrum bacteriostatic activity in Gram-negative and Gram-positive pathogens. Today, there are 61 tetracycline resistance genes sequenced, which are often located on mobile elements and encode for three major mechanisms of resistance: i) tetracycline efflux, ii) ribosomal protection, and iii) tetracycline modification (Thaker M. et al., CMLS 2010, 67(3), 419-31).
i) Tetracycline efflux proteins belong to the major facilitator superfamily and are found in both Gram-positive and Gram-negative bacteria. According to their sequence homology, the efflux proteins are divided into 5 groups: Group 1: Tet(A), Tet(B), Tet(C), Tet(D), Tet(E), Tet(G), Tet(H), Tet(J), Tet(Z), and Tet(30); Group 2: Tet(K) and Tet(L); Group 3: Otr(B) and Tcr(3); Group 4: TetA(P); Group 5: Tet(V); and the non-classified Tet(31), Tet(33), Tet(V), Tet(Y), Tet(34), and Tet(35).
ii) Ribosomal protection proteins share homology with the elongation factors EF-Tu and EF-G and cause a change in conformation of the ribosome after binding, thus preventing tetracycline binding. Members of ribosomal protection proteins are: Tet(M), Tet(0), Tet(S), Tet(W), Tet(32), Tet(36), Tet(Q), Tet(T), Otr(A), and TetB(P).
iii) Tetracycline modification proteins include the enzymes Tet(37) and Tet(X), both of which inactivate tetracycline.
The expression of several of these tet genes is controlled by a family of tetracycline transcriptional regulators known as TetR. TetR family regulators are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity (Ramos J. L. et al., Microbiology and Molecular Biology Reviews 2005, 69(2), 326-356). The TetR proteins identified in over 115 genera of bacteria and archaea share a common helix-turn-helix (HTH) structure in their DNA-binding domain. However, TetR proteins can work in different ways: they can bind a target operator directly to exert their effect (e.g. TetR binds Tet(A) gene to repress it in the absence of tetracycline), or they can be involved in complex regulatory cascades in which the TetR protein can either be modulated by another regulator, or TetR can trigger the cellular response. Most of the defense mechanisms have only transient benefit that is coupled to an associated biological cost. Thus, resistance gene expression is often tightly regulated at the transcriptional or translational level and often triggered by the antibiotic itself.
In the absence of the antibiotic (or antibiotic induced stress signals), the repressor protein is bound to its cognate operator sequence and therefore repressing the transcription of resistance genes. Addition of the antibiotic removes the transcriptional block with subsequent expression of resistance genes.
A Transcriptional Regulator Inhibitory Compound (TRIC) is predicated on the identification of small inhibitory compounds that efficiently block the function of bacterial resistance genes at the highest-possible level, i.e. their transcription. At this point, in general two different interventions exist: (i) inhibitory binding of an activator to its cognate sequence and (ii) constant binding of a repressor to the DNA. Most of the bacterial resistance pathways follow the repressor model (ii), where activation of resistance genes only occurs when the repressor is removed from the DNA.